The construction of a fusion protein involves the linking of two proteins or domains of proteins by a peptide linker. Selection of an appropriate linker sequence is important, as it can affect the function and physical properties of the resulting fusion protein. Often flexible and hydrophilic linkers are chosen so as to not overly constrain and thereby disturb the functions of the domains. The linkers can be used to control the distance and the orientation of the domains. Fusion of a bioactive protein or peptide often results in loss of bioactivity, likely due to steric interference of the fusion partner. Additionally, in the case of Fc fusions, due to their dimeric nature, interference can also occur between the two copies of the heterologous protein.
Mucin proteins and mucin-domains of proteins contain a high degree of glycosylation which structurally allows mucin proteins and other polypeptides comprising mucin domains to behave as stiffened random coils. The present invention is based, in part, on the discovery that this stiffened random coiled structure in combination with the hydrophilic branched hydrophilic carbohydrates that make up the heavily glycosylated mucin domains is particularly useful as a linker in a fusion protein. The rod-like nature of the mucin domains can rigidly separate the bioactive protein from the fusion partner, and thereby be less susceptible to loss in activity. In the case of Fc fusions, the rigid projection away from the Fc will result in greater separation between each copy of the protein of interest, also enabling for larger fusion proteins to be expressed as Fc fusions. Also because of the high level of glycosylation, addition of a mucin domain has the potential to modify the physicochemical properties of a protein such as charge, solubility and viscoelastic properties of concentrated solutions of the active protein.